Toxoplasma gondii is an obligate intracellular parasite that causes severe central nervous system disorders in immunocompromised individuals and birth defects in congenitally infected neonates. Intracellular survival of these organisms is dependent on the ability to invade their host cell, establish a replication-permissive niche, and avoid host cell defenses. The secretory rhoptries have emerged as a key organelle that regulates both invasion and hijacking of host cell functions. For hijacking host functions, the rhoptries inject a burst of proteins into the host cell cytoplasm that can be trafficked to distinct compartments within the infected cell and directly target host pathways. The importance of these host effector proteins is exemplified by a family of rhoptry kinases that are injected and play critical roles in modulating host signaling pathways and regulating parasite virulence. In addition to ROP kinases, the rhoptries inject an array of other effector proteins that are of unknown function. Most of these proteins lack homology to known proteins or identifiable domains and are unique to Toxoplasma and closely related parasites. We have identified a panel of these novel rhoptry effectors and developed gene knockouts in the biologically relevant type II strain parasites for their study. In this proposal, we will determine the role of these novel effector proteins in regulating host functions. Specifically, we will determine the host response to ?effector strains in vitro using host microarray analysis and in vivo by assessing virulence, tissue tropism, and bradyzoite formation in mice. We will additionally use exogenous expression of tandem affinity tagged effector proteins in host cells to determine their destination and identify host targets. Together, these complementary approaches promise to reveal novel mechanistic insights into how Toxoplasma uses this unique set of effector proteins to modulate its mammalian host cell. PUBLIC HEALTH RELEVANCE: Toxoplasma gondii is an intracellular parasite that infects up to a third of the world's human population and causes serious disease in immunocompromised (AIDS) patients and congenitally-infected neonates. Toxoplasma secretes an array of factors from an organelle named the rhoptries that enable it to invade and hijack its host cell. This project is focused determining the function of a novel group of rhoptry effector proteins by examining the host response to gene knockout parasites and identifying host proteins that interact with these novel effectors.